Highly packed polycationic ammonium, sulfonium and phosphonium lipids

ABSTRACT

The present invention discloses highly packed polycationic ammonium, sulfonium and phosphonium lipid compounds useful for making lipid aggregates for delivery of macromolecules and other compounds into cells. They are especially useful for the DNA-dependent transformation of cells. Methods for their preparation and use as intracellular delivery agents are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a division of U.S. patent application Ser.No. 09/648,492, filed Aug. 25, 2000, now allowed, which, in turn, was adivision of U.S. patent application Ser. No. 09/187,676, filed Nov. 6,1998, now U.S. Pat. No. 6,110,916, which, in turn, was a division ofU.S. patent application Ser. No. 08/782,783, now U.S. Pat. No.5,834,439, which, in turn, was a division of U.S. patent applicationSer. No. 08/171,232, filed Dec. 20, 1993, now U.S. Pat. No. 5,674,908,all of which are incorporated by reference herein to the extent notinconsistent herewith.

FIELD OF THE INVENTION

[0002] Highly packed polycationic ammonium, sulfonium and phosphoniumlipid compounds are disclosed, having utility in lipid aggregates fordelivery of macromolecules and other compounds into cells.

BACKGROUND OF THE INVENTION

[0003] Lipid aggregates such as liposomes have been found to be usefulas agents for delivery to introduce macromolecules, such as DNA, RNA,protein, and small chemical compounds such as pharmaceuticals, to cells.In particular, lipid aggregates comprising cationic lipid componentshave been shown to be especially effective for delivering anionicmolecules to cells. In part, the effectiveness of cationic lipids isthought to result from enhanced affinity for cells, many of which bear anet negative charge. Also in part, the net positive charge on lipidaggregates comprising a cationic lipid enables the aggregate to bindpolyanions, such as nucleic acids. Lipid aggregates containing DNA areknown to be effective agents for efficient transfection of target cells.

[0004] The structure of various types of lipid aggregates varies,depending on composition and method of forming the aggregate. Suchaggregates include liposomes, unilamellar vesicles, multilamellarvesicles, micelles and the like, having particle sizes in the nanometerto micrometer range. Methods of making lipid aggregates are by nowwell-known in the art. The main drawback to use of conventionalphospholipid-containing liposomes for delivery is that the material tobe delivered must be encapsulated and the liposome composition has a netnegative charge which is not attracted to the negatively charged cellsurface. By combining cationic lipid compounds with a phospholipid,positively charged vesicles and other types of lipid aggregates can bindDNA, which is negatively charged, can be taken up by target cells, andcan transfect target cells. (Feigner, P. L. et al. (1987) Proc. Natl.Acad. Sci. USA 84:7413-7417; Eppstein, D. et al., U.S. Pat. No.4,897,355.)

[0005] A well-known cationic lipid disclosed in the prior art isN-[1-(2,3-dioleoyloxy) propyl]-N,N,N-trimethylammonium chloride (DOTMA).The structure of DOTMA is:

[0006] DOTMA by itself or in 1:1 combination withdioleoylphosphatidylethanolamine (DOPE) is formulated into liposomesusing standard techniques. Feigner, et al. supra demonstrated that suchliposomes provided efficient delivery of nucleic acids to some types ofcells. A DOTMA:DOPE (1:1) formulation is sold under the trade nameLIPOFECTIN (Gibco/BRL:Life Technologies, Inc., Gaithersburg, Md.).Another commercially available cationic lipid is1,2-bis(oleoyloxy)-3-3-(trimethylammonia) propane (DOTAP), which differsfrom DOTMA only in that the oleoyl moieties are linked via ester, ratherthan ether bonds to the propylamine. DOTAP is believed to be morereadily degraded by target cells. A related group of prior art compoundsdiffer from DOTMA and DOTAP in that one of the methyl groups of thetrimethyl-ammonium group is replaced by a hydroxyethyl group. Compoundsof this type are similar to the Rosenthal Inhibitor (RI) ofphospholipase A (Rosenthal, A. F. and Geyer, R. P. (1960) J. Biol. Chem.235:2202-2206) which has stearoyl esters linked to the propylamine core.The dioleoyl analogs of RI are commonly abbreviated as DORI-ether andDORI-ester, depending on the linkage of the fatty acid moieties to thepropylamine core. The hydroxy group can be used as a site for furtherfunctionalization, for example by esterification to carboxyspermine.

[0007] Another class of prior art compounds has been disclosed by Behret al. (1989) Proc. Natl. Acad. Sci. USA 86:6982-6986; EPO publication 0394 111 (Oct. 24, 1990), in which carboxyspermine has been conjugated totwo types of lipids. The structure of 5-carboxyspermyl-glycinedioctadecylamide (DOGS) is:

[0008] where R=CH₃(CH₂)₁₇

[0009] The structure of dipalmitoylphosphatidylethanolamine5-carboxyspermylamide (DPPES) is:

[0010] where R=CH₃(CH ₂)₁₅

[0011] Both DOGS and DPPES have been used to coat plasmids, forming alipid aggregate complex that provides efficient transfection. Thecompounds are claimed to be more efficient and less toxic than DOTMA fortransfection of some cell lines. DOGS is available commercially asTRANSFECTAM™ (Promega, Madison, Wis.).

[0012] A cationic cholesterol derivative (DC-Chol) has been synthesizedand formulated into liposomes in combination with DOPE. (Gao, X. andHuang, L. (1991) Biochim. Biophys. Res. Comm. 179:280-285) Thecompound's structure is

[0013] Liposomes formulated with DC-Chol are said to provide moreefficient transfection and lower toxicity than DOTMA-containingliposomes for some cell lines.

[0014] Lipopolylysine, formed by conjugating polylysine to DOPE, hasbeen reported to be especially effective for transfection in thepresence of serum, a condition likely to be encountered in vivo (Zhou,X. et al. (1991) Biochim. Biophys. Acta 1065:8-14).

[0015] Despite advances in the field, a need remains for a variety ofimproved cationic lipid compounds. In particular, no single cationiclipid to date has been found to work well with all cell types. Sincedifferent cell types differ from one another in membrane composition, itis not surprising that different compositions and types of lipidaggregates are effective for different cell types, either for theirability to contact and fuse with target cell membranes, or for aspectsof the transfer process itself. At present these processes are not wellunderstood, consequently the design of effective liposomal precursors islargely empirical. Besides content and transfer, other factors are ofimportance, for example, ability to form lipid aggregates suited to theintended purpose, toxicity to the target cell, stability as a carrierfor the compound to be delivered, and ability to function in an in vivoenvironment. In addition, lipid aggregates can be improved by broadeningthe range of substances which can be delivered to cells. The highlypacked and spatially correct positioning of positive charges matchingnegative charges on DNA polycationic ammonium, sulfonium and phosphoniumlipid compounds of the present invention have improved function withrespect to several of the foregoing attributes.

SUMMARY OF THE INVENTION

[0016] The present invention provides highly packed polycationicammonium, sulfonium and phosphonium lipid compounds according to thegeneral formula:

[0017] In the general formula (I),

[0018] X is selected from the group consisting of N, S, P or SO;

[0019] x is an integer ranging from 1 to about 20;

[0020] n₁, where i=1 to x, are, independently of one another, integersthat can have a value ranging from 1 to about 6;

[0021] R_(A) and R_(B), independently of one another, are selected fromthe group consisting of H, or an alkyl, hydroxyalkyl or thiolsubstituted alkyl group having from 1 to about 6 carbon atoms;

[0022] R₁ and R₂, independently of one another, are selected from thegroup consisting of alkyl groups having from 1 to about 6 carbon atoms,where r is either 1 or 0, such that r is 0 or 1 when X is N, with Nbeing positively charged if r is 1, r is 0 when X is S or SO, with S andSO being positively charged, and r is 1 when X is P, with P beingpositively charged; and

[0023] A₁-A₂, independently of one another, are selected from the groupconsisting of the following groups Z₁-Z₆:

[0024] Z₁ is a straight-chain alkyl, alkenyl, or alkynyl group havingfrom 2 to about 22 carbon atoms wherein one or more non-neighboring—CH₂— groups can be replaced with an O or S atom;

[0025] Z₂ is a branched alkyl, alkenyl, or alkynyl group having from 2to about 22 carbon atoms wherein one or more non-neighboring —CH₂—groups can be replaced with an O or S atom;

[0026] Z₃ is a straight-chain or branched alkyl group substituted withone or two OH, SH, NH₂ or amine groups within about 3 carbon atoms ofthe bond between Z₃ and X;

[0027] Z₄ is a substituted straight-chain or branched alkyl, alkenyl oralkynyl group having from 2 to about 22 carbon atoms wherein thesubstituent is an aromatic, alicyclic, heterocyclic or polycyclic ringand wherein one or more of the non-neighboring —CH₂— groups of saidalkyl, alkenyl or alkynyl group can be substituted with an O or S atom.

[0028] Z₅ is a —B—L group wherein B is selected from the group —CO—,—CO₂—, —OCO—, —CO—N—, —O—CO—N—, —O—CH₂—, —CH₂—O—, —S—CH₂—, —CH₂—S— or—CH₂— and L is selected from the group consisting of:

[0029] Z₁; Z₂; Z₄; or

[0030] an aromatic, alicyclic, heterocyclic or polycyclic ring moiety;

[0031] Z₆ is a —CH(D—L)₂ or a —C(D—L)₃ group wherein D is selected fromthe group consisting of —CO—, —CO₂—, —OCO—, —CO—N—,

[0032] —O—CO—N—, —O—, or —S— and L is selected from the group consistingof:

[0033] Z₁; Z₂; Z₄; or

[0034] an aromatic, alicyclic, heterocyclic or polycyclic ring moiety.

[0035] In general in any particular compound of formula I, the chainlength n₁ can vary from 1 to 6. For example, in a compound in which x is3 where i is 1 to 3, n₁, n₂ and n₃ can all have the same value, any twocan have the same value or all three can have different values. Inparticular embodiments of the compounds of this invention, the chainlengths n₁ vary in the repeating pattern 3, 4, 3, 3, 4, 3, . . . Otherparticular embodiments of this invention include those in which n_(i)differ from each other by +/−1.

[0036] A groups include those in which two substituents on differentX's, preferably neighboring X groups, are covalently linked with eachother to form a cyclic moiety.

[0037] The oxygen or sulfur atoms introduced into Z₁ and Z₂ groups arepreferably introduced within about 3 carbon atoms from the bond to the Xgroup.

[0038] The aromatic, alicyclic, heterocyclic or polycyclic ring moietiesof this invention can be substituted or unsubstituted. Substituentsinclude among others: OH, SH, NH₂,CH₃, COCH₃ and halogens, particularlyF. Further, one or more of the ring carbons in the alicyclic,heterocyclic or polycyclic ring moieties of this invention can becarbonyl groups C═O.

[0039] Introduction of OH, NH₂ or amine groups as substituents on Agroups within about 3 carbons from the bond to X can facilitatesolubility of the compounds of this invention in physiological media.

[0040] Compounds of the invention are useful, either alone or incombination with other lipid aggregate-forming components (e.g., DOPE,DOSPA, DOTMA or cholesterol) for formulation into liposomes or otherlipid aggregates. Such aggregates are polycationic, able to form stablecomplexes with anionic macromolecules, such as nucleic acids. Thepolyanion-lipid complex interacts with cells making the polyanionicmacromolecule available for absorption and uptake by the cell.

[0041] Of special interest are the products of general formula (I) inwhich X is nitrogen and A₁ and A₂ are Z₁. These N-alkylated-polyaminesand their quaternary ammonium salts are particularly useful forintracellular delivery of negatively charged macromolecules. This aspectof the invention is based on the finding that polyamines alkylated withlong hydrocarbon chains have enhanced affinity for cells, many of whichbear a net negative charge, and for various polyanions, such as nucleicacids, relative to the natural polyamine compounds. The effectiveness ofN-alkylated polyamines is thought to result from the increased basicity(easier and stronger protonation) of the secondary and tertiary amines,even under alkaline conditions. Similarly, quarternized polyaminecompounds show increased affinity for negatively charged substances,such as cells and nucleic acids, relative to the natural polyamines. Theincrease in affinity of the quarternized polyamine compounds presumablyresults from the permanent positive charges of the quarternized amines,each aligned in front of a negative charge of the DNA molecule(cooperative effect). Moreover, because of the relatively high lipidcontent of the alkylated and quarternized polyamines, these compoundsalso interact more strongly with the lipid bilayer of cell membranesthan their cognate polyamines.

[0042] The combination of high lipid content per molecule and increasedaffinity for anionic macromolecules makes the compounds of the inventionnot only superior intracellular delivery agents, but also less toxic tothe target cells. The reduced toxicity is thought to result in part fromthe fact that a higher binding constant between DNA/lipid leads to alower concentration of lipid needed to completely coat the DNA. Sincetoo much lipid disrupts the cell membrane (these lipids are alsodetergents), a lower amount of lipid should be less toxic. Thisincreased affinity produces a more stable liposome, which in turnincreases the efficiency of delivery. Thus, lower concentrations ofthese highly packed polycationic lipids are required to coat themolecules and bind to the target cells, thereby maximizing efficiency ofdelivery while minimizing cell toxicity.

[0043] In addition to the high lipid content and increased affinity foranionic substances, the compounds of the present invention also promoteproximity between the complexed polyanion and the target cell membrane,thus increasing interactions between these two entities. In mostliposomal precursors, the lipid moiety is physically separated from thecationic binding site by a relatively large linking group, typicallyranging from five to eight carbon atoms in length. The presentinvention, in contrast, provides straight-chain compounds wherein thelipid constituent is directly attached, without a linker, to thecationic site. The branched compounds of the invention comprise a short(preferably 3 carbon) linker, which enhances the solubility of thesehighly packed lipids. It is believed that the unexpected improvement inefficiency and cell viability observed with these compounds can beattributed, at least in part, to proximity between the liposomalcontents and cell membrane. Although the mechanism is not fullyunderstood, it is believed that less water can intercalate in thisregion, thus minimizing disruption to the cell membrane caused by excesswater.

[0044] Of particular interest are the products of general formula (I) inwhich X is nitrogen, n is 3 or 4, x is 1, r is 0, R_(A) and R_(B) arehydrogens, and A₁ and A₂ are unbranched alkyl, alkenyl, alkynyl oralkoxy groups having 2 to about 22 carbon atoms.

[0045] Most preferred is tetramethyltetrapalmylspermine, the product ofgeneral formula (I) in which X is nitrogen, where x is 3, n₁ is 3, n₂ is4, and n₃ is 3, R_(A) and R_(B) are hydrogens, R₁ and R₂ are methylgroups, and A₁ and A₂ are unbranched alkyls having 16 carbon atoms.

[0046] Specific embodiments of this invention include compounds offormula I in which X is N. Of those compounds in which X is N, thisinvention includes, but is not limited to those compounds wherein:

[0047] A₁ and A₂, independently of one another, are selected from thegroup Z₁, Z₂, or Z_(3;)

[0048] A₁ and A₂, independently of one another, are selected from thegroup Z₅; and particularly those Z₅ wherein B is CO, —CH₂—, or —O—CH₂—and L is Z₁ or Z_(2;)

[0049] A₁ and A₂, independently of one another, are selected from thegroup Z₆; and particularly wherein D is —O—, —CO—, —OCO—or —CO₂—;

[0050] R_(A) and R_(B) are H or they are alkyl groups having 1-3 carbonatoms, inclusive;

[0051] R₁ and R₂ are alkyl groups having 1 to 3 carbon atoms, inclusive,and more particularly are methyl groups;

[0052] n_(i) are all either 3 or 4 and x is 2 to 5;

[0053] n_(i) alternate in the pattern 3, 4, 3, 3, 4, 3 and x is greaterthan or equal to 3; and

[0054] R_(A) and R_(B) are —CH₂—CH₂—OH groups.

[0055] Compounds of this invention of formula I in which X is S includeamong others those in which:

[0056] A₁ and A₂ are R groups which are selected from any of R₅-R₈where:

[0057] R5 is a straight-chain (unbranched) alkyl, alkenyl, alkynyl oralkoxy having 2 to about 22 carbon atoms;

[0058] R6 is a branched alkyl, alkenyl, alkynyl or alkoxy having 2 toabout 22 carbon atoms;

[0059] R₇ is an aromatic, alicyclic, heterocyclic or polycyclic ringmoiety; and

[0060] R₈ is a branched or unbranched substituted alkyl, alkenyl,alkynyl or alkoxy having from 2 to about 22 carbon atoms, wherein thesubstituent is an aromatic, alicyclic, heterocyclic or polycyclic ring.

[0061] Also included in this invention are compounds of formula I whereX is S and x is 1. In this case, R_(A) and R_(B) are preferably methylgroups. Preferred A groups having alkyl, alkenyl alkynyl or alkoxygroups are those having about 12 to 16 carbon atoms. Preferred R arebranched or straight-chain alkyl, alkenyl or alkynyl groups. In R₅, R₆,and the branched or straight-chain portion of R₈, one or morenon-neighboring —CH₂— groups can be replaced with O or S atoms to giveether or thioether R groups.

[0062] Additional subsets of compounds of formula I of this inventioninclude those in which:

[0063] x=1, n is an integer between 2 and 6 inclusive and R is anunbranched alkyl, alkenyl, alkynyl or alkoxy group having 2 to about 22carbon atoms; and particularly those in which n is 3;

[0064] Wherein at least two of the R groups on different S arecovalently linked together to produce a cyclic moiety.

[0065] In specific embodiments, the compounds of the present inventionare also represented by the formulas II and III:

[0066] In both formula II and III, R_(A), R_(B), r, Z₁-Z₆ and X are asdefined above for formula I. All of R₁-R₄ can be selected from thegroups as defined above for R₁ and R₂.

[0067] In formula II:

[0068] n and m, independently of one another, are integers (chainlength) ranging in value from 1 to about 6, with n and m of 3 or 4 beingmore preferred. It is preferred that the values of m and n differ onlyby +/−1;

[0069] x can be an integer from 1 to 10, with the subset of compoundshaving x=2-5 being of particular interest; and

[0070] A₁-A₃, independently of one another, are selected from the groupZ₁-Z₆, of particular interest are A groups which are straight-chainalkyl, alkenyl or alkynyl groups.

[0071] In formula III:

[0072] l, m, and n, independently of one another, are integers (chainlength) ranging in value from 1 to about 6, with l, m and n of 3 or 4being more preferred. It is preferred that the values of l, m and ndiffer from each other only by +/−1;

[0073] x can be an integer from 1 to 10, with the subsets of compoundshaving x=1, x=2 and x=3-5 being of particular interest; and

[0074] A₁-A₄, independently of one another, are selected from the groupZ₁-Z₆, with A groups which are straight-chain alkyl alkenyl or alkynylgroups of particular interest.

[0075] Specific embodiments of this invention include compounds offormulas II and III in which X is N, m is 3, and n is 4, wherein A₁-A₃,independently of one another, are selected from the groups Z₁, Z₂, andZ₃, and wherein A₁-A₃ are the same group. Other specific embodimentsinclude compounds of formulas II and II in which X is N and A₁-A₃,independently of one another, are selected from the group Z₅, wherein Bis CO, —CH₂—, or —O—CH₂— and L is Z₁ or Z₂. Other embodiments includecompounds of formulas II and III in which X is N and A₁-A₄,independently of one another, are selected from the group Z₆, wherein Dis —O—, —CO—, —OCO— or —CO₂—. Of particular interest are compounds offormulas II and III wherein X is N and R_(A) and R_(B) are H or methylgroups, R₁ and R₂ are methyl groups, m is 3, n is 4, x is 2 to 6. Alsoof particular interest are compounds of formulas II and III wherein X isN and R_(A) and R_(B) are —CH₂—CH₂—OH groups.

[0076] Of particular interest are compounds of formula III in which X isN; R_(A) and R_(B) are H; l, m and n are 3 or 4; x is 1; and A₁-A₄,independently of one another, are unbranched alkyl groups having fromabout 12 to 16 carbon atoms. Particularly preferred are compounds offormula III where A₁-A₄ are all the same unbranched alkyl group,particularly an unbranched alkyl group having 16 carbon atoms. Otherembodiments include compounds of formula III wherein X is N; x is 1; l,m and n are 3 or 4; and A₁-A₄ is Z₅, wherein B is CO and L is Z₁,particularly wherein Z₁ has about 12 to about 16 carbon atoms. Otherspecific embodiments include the compounds of formula III wherein X isS; x is 1-5; l, m and n are 2 or 3; and A₁-A₄ is Z₁, particularlywherein A₁-A₄ are unbranched alkyl or alkenyl groups having from 2 toabout 22 carbon atoms, and preferably from about 12 to about 16 carbonatoms. Also of interest are compounds of formula III wherein at leasttwo of the A₁-A₄ are substituents are covalently linked to produce acyclic compound. Other specific embodiments include the compounds offormula III wherein X is P.

[0077] In compounds of both formula II and III, preferred Z groups arebranched or straight-chain alkyl, alkenyl, alkynyl or alkoxy groups,have about 12 to about 16 carbon atoms.

[0078] This invention also includes compounds having the structure:

[0079] wherein Ar₁ and Ar₂ are aryl rings or cyclohexane rings and R isselected from any of R₅- R₈ as defined above. The aryl rings can beselected from among any of phenyl, pyridinyl, pyrimidyl, pyrazinyl,thiadiazole and pyridazinyl. Cyclohexane rings include trans-cyclohexylrings. Of particular interest are compounds of formula IV wherein bothof Ar₁ and Ar₂ are phenyl rings, and wherein R is R₅ or an unbranchedalkyl.

[0080] This invention also includes lipid aggregates comprising one ormore of the compounds of formulas I, II, III or IV or mixtures thereofOf particular interest are lipid aggregates of the compounds of formulaI, more particularly those in which X is N.

[0081] The transfection methods of the present invention employingcompounds of formulas I, II, III or IV or mixtures thereof can beapplied to in vitro and in vivo transfection of cells, particularly totransfection of eukaryotic cells including animal cells. The methods ofthis invention can be used to generate transfected cells which expressuseful gene products. The methods of this invention can also be employedas a step in the production of transgenic animals. The methods of thisinvention are useful as a step in any therapeutic method requiringintroducing of nucleic acids into cells. In particular, these methodsare useful in cancer treatment, in in vivo and ex vivo gene therapy, andin diagnostic methods. The transfection compositions of this inventioncan be employed as research reagents in any transfection of cells donefor research purposes. Nucleic acids that can be transfected by themethods of this invention include DNA and RNA from any source comprisingnatural bases or non-natural bases, and include those encoding andcapable of expressing therapeutic or otherwise useful proteins in cells,those which inhibit undesired expression of nucleic acids in cells,those which inhibit undesired enzymatic activity or activate desiredenzymes, those which catalyze reactions (Ribozymes), and those whichfunction in diagnostic assays.

[0082] The compositions and methods provided herein can also be readilyadapted in view of the disclosure herein to introduce biologicallyactive anionic macromolecules other than nucleic acids including, amongothers, polyamines, polyamine acids, polypeptides, proteins, biotin, andpolysaccharides into cells. Other materials useful, for example astherapeutic agents, diagnostic materials and research reagents, can becomplexed by the polycationic lipid aggregates and introduced into cellsby the methods of this invention.

[0083] This invention also includes transfection kits which include oneor more of the compounds of formulas I, II, III, IV or mixtures thereofas cationic lipids.

DETAILED DESCRIPTION OF THE INVENTION

[0084] The present invention provides novel, highly packed polycationicammonium, sulfonium and phosphonium lipid compounds having uniqueproperties and advantages not heretofore available to the liposome art.The compounds can be used alone or in combination with other compounds(e.g., DOPE, DOTMA and DOSPA) to prepare liposomes and other lipidaggregates suitable for transfection or delivery of compounds other thanDNA to target cells, either in vitro or in vivo.

[0085] The novel compounds of general formula (I) are polycationic andthus form highly stable complexes with various anionic macromolecules,particularly polyanions such as nucleic acids. These compounds have theproperty, when dispersed in water, of forming lipid aggregates whichassociate strongly, via their cationic portions, with polyanions. Byusing an excess of cationic charges relative to the anionic compound,the polyanion-lipid complexes may be adsorbed on cell membranes, therebyfacilitating uptake of the desired compound by the cells.

[0086] The cationic lipids disclosed herein offer three uniqueadvantages over prior art compounds. First, the compounds of generalformula (I) represent novel liposomal precursors wherein thepolycationic binding regions are optimally spaced, preferablyequidistance between charges, to provide proper alignment with theanionic phosphates of nucleic acids. Proper alignment of chargesincreases the binding constant of the lipid to nucleic acid viacooperative interaction. This increased affinity produces a more stableDNA-lipid complex, which in turn increases the efficiency of delivery.Thus, lower concentrations of these agents are required to coat themolecules and bind to the target cells, thereby maximizing efficiency ofdelivery while minimizing cell toxicity.

[0087] The second unique advantage of the compounds disclosed herein istheir unusually high affinity for the lipid bilayer of cell membranes.Unlike the mono-substituted lipopolyamine compounds currently in use,the compounds of the invention comprise lipidic substituents at eachcationic binding region. Compounds of the invention thus interact morestrongly with the lipid bilayer of cell membranes than existing cationiclipids. The branched lipids of the present invention comprise multiplelipid moieties per cationic site, and thus bind particularly strongly tocell membranes.

[0088] Finally, the compounds of the present invention promote proximitybetween the complexed polyanion and the target cell membrane, thusincreasing interactions between these two entities. Unlike previouscationic lipids, the lipid moieties of the present compounds areattached without linkers to the cationic sites.

[0089] Of special interest are the products of general formula (I) inwhich X is nitrogen and A₁ and A₂ are Z₁. These N-alkylated-polyaminesand their quaternary ammonium salts are especially useful forintracellular delivery of negatively charged macromolecules. Theunexpected effectiveness is thought to result from the increasedbasicity and the permanent positive charge of the alkylated andquarternized polyamines, respectively. Moreover, as discussed elsewhereherein, the relatively high lipid content of these compounds is believedto maximize interaction with the lipid bilayer of cell membranes. Thecombination of high lipid content and increased affinity for anionicmacromolecules makes these compounds not only superior intracellulardelivery agents, but also less toxic to the target cells.

[0090] In a preferred embodiment of general formula (I), the values ofn₁ are either identical or vary at most by one integer, thus providingapproximately equidistant spacing between the polycationic bindingregions (X). In another preferred embodiment of general formula (I),when A₁ and A₂ are branched hydrocarbons (e.g., Z₂), each branch isconnected to the polymer backbone via a hydrophilic heteroatom such asoxygen, as exemplified herein. The presence of a hydrophilic heteroatommitigates the hydrophobicity of these highly packed compounds, thusenhancing their solubility in water.

[0091] Certain of the compounds of this invention may be insufficientlysoluble in physiological media to employ for delivery and transfectionmethods. Those of ordinary skill in the art will appreciate that thereare a variety of techniques available in the art to enhance solubilityof such compounds in aqueous media. Such methods are readily applicablewithout undue experimentation to the compounds described herein. Asdescribed herein, one method for increasing solubility of compounds ofFormulas I through III is to introduce OH, NH₂, SH, or aminesubstituents on Z groups within about 3 carbon atoms from the X group.

[0092] The present invention also provides improved methods fortransfecting and delivering macromolecules to target cells. Theimprovement relates to the use of highly packed polycationic ammonium,sulfonium and phosphonium lipid compounds to either enhance theefficiency of delivery or to reduce the toxicity to the cells. Thisinvention has significant advantages over prior art methods which employneutral or slightly basic delivery agents having a relatively low lipidcontent, which interact weakly with both anionic macromolecules and thelipid bilayer of cell membranes. Because of this limited affinity andlow lipid content, current methods require high concentrations of thedelivery agent, which disrupts cell membranes, often leading to celldeath. The present invention resolves the problems associated with priorart methods by employing highly efficient delivery agents which areeffective at relatively low and non-toxic concentrations.

DEFINITIONS

[0093] Lipid Aggregate is a generic term which includes liposomes of alltypes both unilamellar and multilamellar as well as micelles and moreamorphous aggregates of cationic lipid or lipid mixed with amphiphaticlipids such as phospholipids.

[0094] Target Cell refers to any cell to which a desired compound isdelivered, using a lipid aggregate as carrier for the desired compound.

[0095] Transfection is used herein to mean the delivery of expressiblenucleic acid to a target cell, such that the target cell is renderedcapable of expressing said nucleic acid. It will be understood that theterm “nucleic acid” includes both DNA and RNA without regard tomolecular weight, and the term “expression” means any manifestation ofthe functional presence of the nucleic acid within the cell, includingwithout limitation, both transient expression and stable expression.

[0096] Delivery is used to denote a process by which a desired compoundis transferred to a target cell such that the desired compound isultimately located inside the target cell or in, or on, the target cellmembrane. In many uses of the compounds of the invention, the desiredcompound is not readily taken up by the target cell or appropriatecytoplasmic compartment and delivery via lipid aggregates is a means forgetting the desired compound into the cell cytoplasm.

[0097] The polycationic lipids were prepared by following the generalreaction schemes given below (Schemes 1-5).

[0098] Straight-chain polycationic lipopolyamines were prepared as shownin Scheme 1. A polyamine was treated with an acid chloride of thedesired length in the presence of triethylamine and methylene chlorideunder argon at room temperature to obtain the corresponding substitutedamide (compound 1). Compound 1 was then reduced using lithium aluminumhydride in the presence of anhydrous tetrahydrofurane to give compound2. Treatment of compound 2 with iodomethane at high temperature yieldeda partially quarternized compound (compound 3). Compound 3 was furthermethylated using additional iodomethane to produce the fullyquarternized spermine derivative (compound 4).

[0099] Although the above method exemplifies the synthesis ofN,N,N′,N′-tetrapalmylspermine and its quarternized derivatives, thereaction scheme provides a general method for preparing a variety ofN-alkylated and quarternized lipopolyamines. Those of ordinary skill inthe art will appreciate that alternate methods and reagents other thosespecifically detailed herein can be employed or readily adapted toproduce a variety of useful compounds. For example, although the abovereaction scheme uses spermine as the exemplified starting material, thescheme is equally applicable with other straight-chain polyamines.Various polyamines are readily accessible or can be easily synthesizedusing standard methods, for example, by combining acrylonitrile with anappropriate diaminoalkane.

[0100] Branched polycationic lipopolyarnines are prepared as shown inScheme 2. A branched amino alcohol is treated with an appropriatenitrile (e.g., acrylonitrile) to produce a branched amino nitrile of thedesired length (compound 4). Compound 4 is then alkylated using3-bromo-acrylonitrile at high temperature to yield the correspondingdinitrile compound (compound 5). Compound 5 is further alkylated usingan alkyl sulfonate in pyrrole to yield compound 6. Reduction of thenitrilo groups of compound 6 using lithium aluminum hydride or,alternatively, using hydrogen in the presence of Raney nickel, yieldscompound 7. Compound 7 is then treated with an acid chloride of thedesired length in the presence of triethylamine and methylene chloride,followed by reduction with lithium aluminum hydride at high temperatureto give compound 8.

[0101] Although the above method uses tri-hydroxymethyl-aminomethane asthe exemplified starting material, the reaction scheme provides ageneral method for preparing a variety of branched lipopolyamines. Thesealkylated polyamines can also be quarternized as previously described(see compounds 3 and 4 above) using iodomethane at high temperature.Those of ordinary skill in the art will appreciate that alternatemethods and reagents can be employed or readily adapted to produce avariety of useful branched compounds.

[0102] Dicationic sulfonium lipids were prepared as shown in Scheme 3.1,3-propanedithiol was treated with ethylene oxide to afford thecorresponding diol-adduct. The diol was tosilated in the presence ofbase followed by displacement of the tosyl groups by sodium sulfide togive the corresponding mercaptan. This mercaptan was di-alkylated withacetyl bromide using N-butyl lithium as the base. Finally, treating thetetra sulfide with iodomethane affords the sulfonium salt. Polycationicsulfonium lipids were prepared as shown in Scheme 4. Schemes 3 and 4provide a general method for preparing a variety of highly packedsulfonium lipids. Modification and optimization of this method toproduce a variety of useful sulfonium compounds is well within the skillof the ordinary artisan.

[0103] Phosphonium lipids were prepared as shown in Scheme 5.Diphenylphosphinic chloride was treated with hydroquinine (excess) toafford the corresponding addition product. Treatment of the additionproduct with sodium hydride followed by acetyl bromide afforded thecorresponding phospholipid. The phospholipid, in turn, is quarternizedwith iodomethane to afford the phosphonium salt.

[0104] The compounds of the invention can be used in the same manner asare prior art compounds such as DOTMA, DOTAP, DOGS and the like. Methodsfor incorporating such cationic lipids into lipid aggregates arewell-known in the art. Representative methods are disclosed by Felgneret al., supra; Eppstein et al. supra; Behr et al. supra; Bangham, A. etal. (1965) M. Mol. Biol. 23:238-252; Olson, F. et al. (1979) Biochim.Biophys. Acta 557:9-23; Szoka, F. et al. (1978) Proc. Natl. Acad. Sci.USA 75:4194-4198; Mayhew, E. et al. (1984) Biochim. Biophys. Acta775:169-175; Kim, S. et al. (1983) Biochim. Biophys. Acta 728:339-348;and Fukunaga, M. et al. (1984) Endocrinol. 115:757-761. Commonly usedtechniques for preparing lipid aggregates of appropriate size for use asdelivery vehicles include sonication and freeze-thaw plus extrusion.See, e.g., Mayer, L. et al. (1986) Biochim. Biophys. Acta 858:161-168.Microfluidization is used when consistently small (50 - 200 nm) andrelatively uniform aggregates are desired (Mayhew, E., supra).Aggregates ranging from about 50 nm to about 200 nm diameter arepreferred; however, both larger and smaller sized aggregates arefunctional.

[0105] Methods of transfection and delivery of other compounds arewell-known in the art. The compounds of the present invention yieldlipid aggregates that can be used in the same processes as those priorart compounds.

[0106] It will be readily apparent to those of ordinary skill in the artthat a number of general parameters are important for optimal efficiencyof transfection or delivery. These parameters include, for example, thepolycationic lipid concentration, the concentration of compound to bedelivered, the medium employed for delivery, the length of time thecells are incubated with the polyanion-lipid complex, and the relativeamounts of cationic and non-cationic lipid. It may be necessary tooptimize these parameters for each particular cell type. Suchoptimization is routine employing the guidance provided herein,including the transfection assays as described in the Examples herein.

[0107] It will also be apparent to those of ordinary skill in the artthat alternative methods, reagents, procedures and techniques other thanthose specifically detailed herein can be employed or readily adapted toproduce the liposomal precursors and transfection compositions of thisinvention. Such alternative methods, reagents, procedures and techniquesare within the spirit and scope of this invention.

[0108] The preparation and use of representative compounds of theinvention are further detailed by reference to the following Examples.In each case, the ability of various compounds of the invention toprovide efficient transfection was compared with a control usingDOSPA:DOPE (1.5:1 molar ratio). All abbreviations used herein arestandard abbreviations in the art. Specific procedures not described indetail are either referenced or well-known in the art.

EXAMPLES Example 1 Synthesis of N,N,N′,N′ -tetrapalmitoylspermine (1)

[0109] To a solution of spermine (0.99 g, 80%,3.8 mmol) andtriethylamine (1.54 g, 15.2 mmol) in methylene chloride (400 ml) at 0°C. under argon was added palmitoyl chloride (4.17 g, 4.8 ml, 15.2 mmol).The reaction continued at room temperature for three days. TLC analysis(silica gel; THF:CH₂Cl₂/1:3) showed a new spot (rf=0.8) and no startingmaterial. The organic phase was washed twice with sodium bicarbonatesolution (10%, 200 ml), hydrochloric acid (1 M, 200 ml) and water (200ml). The solution was dried (Na₂SO₄) and the solvent removed to afford 4g of desired product (90%).

Example 2 Synthesis of N,N,N′N′-tetrapalmylspermine (2)

[0110] To a suspension of lithium aluminum hydride (900 mg, 23.7 mmol)in anhydrous tetrahydrofurane (80 ml) was added a solution ofN,N,N′,N′-tetrapalmitoylspermine (500 mg, 0.43 mmol) in anhydroustetrahydrofurane (10 ml). The reaction mixture was refluxed for two daysunder argon. The excess lithium aluminum hydride was removed with sodiumhydroxide (1 M, 5 ml). The organic phase was decanted and the flaskwashed twice with additional tetrahydrofurane (50 ml). The solution wasdried (Na2SO4) and the solvent removed in vacuo to afford almost puredesired tetraamine. This material was passed through a short-path silicagel bed (filtration) and eluted sequentially with ethyl acetate andethyl acetate/triethylamine (10%) to afford, after solvent removal, thedesired product (313 mg, 82%).

Example 3 Synthesis ofN,N,N′,N′-tetramethyltetrapalmyl-sperminetetrammonium iodide (3)

[0111] A solution of tetrapalmylspermine (20 mg) in iodomethane (1 ml)was heated at 55° C. for 18 hr. The excess iodomethane was removed invacuo and the residue redissolved in methylene chloride. This solutionwas extracted twice with sodium bicarbonate (3 ml), dried (NaSO₄), andthe solvent removed to afford 25 mg (100%) of desired material.

Example 4 Synthesis ofN,N,N′,N′-hexamethyltetrapalmyl-sperminetetrammonium iodide (4)

[0112] A solution ofN,N,N′,N′-tetramethyltetrapalmylspermine-tetrammonium iodide (10 mg) iniodomethane was heated for 48 hr at 80° C. The iodomethane was removedin vacuo to afford 12 mg of desired product (100%).

Example 5 Lipid formulation

[0113] Tetrapalmylspernine, tetramethylpalmyl-spermine, andhexamethylpalmylspennine were formulated using two protocols. Lipidswere first dissolved in chloroform and aliquots of each lipid with andwithout DOPE were evaporated under vacuum. Lipids were then resuspendeddirectly in water by vortexing, or were suspended first in ethanol({fraction (1/10)} of final volume) and then diluted with water.

Example 6 Cell culture and plasmids

[0114] All cell lines were obtained from American Type CultureCollection (Rockville, Md.). Standard tissue culture methods wereemployed. Chinese hamster ovary (CHO-K1) cells were cultured in EMEM(GIBCO BRL) containing 2 mM proline and 5% (v/v) fetal bovine serum(FBS). NIH 3T3 cells were cultured in Dulbecco's-modified Eagle's medium(DMEM) containing 10% (v/v) calf serum (CS). Jurkat cells were culturedin RPMI 1640 (GIBCO BRL) containing 10% (v/v) FBS. Human fibroblastswere isolated from neonatal foreskin tissue and prepared as follows.Rinsed, fresh tissue was exposed to 25 units/ml dispase (CollaborativeResearch, Bedford, Mass.) overnight at 4° C., separated into epidermisand dermis, and the minced dermis was digested 10 min at 37° C. in 0.25%(v/v) trypsin, 1 mM EDTA. The reaction was stopped by a rinse andcentrifugation in DMEM with 10% (v/v) FBS, in which the cells were alsocultured. All cultures were incubated at 37° C, 5% CO₂. Media for allcultures routinely included 100 units/ml penicillin and 100 μg/mlstreptomycin.

[0115] The plasmid vector pCMV β-gal is a commercially available(Clontech, Calif.) mammalian reporter vector containing the E. coliβ-galactosidase (β-gal) gene under the control of the Cytomegaloviruspromoter (see MacGregor et al. (1989) Nucleic Acids Res. 17:2365). Theplasmid vector pCMVCAT was also described previously (Boshart et al.(1985) Cell 41:521). Plasmid DNA was purified by standard cesiumchloride methods.

Example 7 Transfection of CHO-K1, NIH-3T3, and human fibroblast cells

[0116] For transfection of CHO-K1, NIH-3T3, and human fibroblast cellsin 24-well plates, lipid and DNA (pCMV β-gal) were diluted separatelyinto 25 μl aliquots of Opti-MEM I Reduced Serum Medium (GIBCO BRL;serum-free). These aliquots were gently mixed and incubated at roomtemperature for 15-45 minutes to form lipid-DNA complexes. The complexeswere added to cells in each well containing 250 μl serum-free growthmedium. Cells were exposed to DNA-lipid complexes for 4-5 hr understandard culture conditions, after which 1 ml normal growth medium wasadded. Antibiotics were never present during lipid-mediatedtransfections. At 24 hr after transfection, cells were assayed in situfor ρ-galactosidase activity.

Example 8 Transfection of Jurkat cells

[0117] For transfection of Jurkat cells in suspension, lipid and DNA(pCMVCAT) were diluted separately into 500 μl aliquots of Opti-MEM IReduced Serum Medium (GIBCO BRL; serum-free). These aliquots were gentlymixed and incubated at room temperature for 15-45 minutes to formlipid-DNA complexes. For each transfection sample, 1×10⁶ Jurkat cellswere centrifuged in a microfuge tube. The cell pellets were suspendedwith the lipid-DNA complex solutions and transferred to wells of 12-wellplates. Cells were exposed to DNA-lipid complexes for 4-5 hr understandard culture conditions, after which 0.5 ml growth medium containing30% FBS, 150 μg/ml Phorbol myrystate acetate (PMA; Sigma Chemical Co.,St. Louis, Mo.), and 3 μg/ml phytohemagglutinin (PHA) were added to afinal concentration of 10% FBS, 50 μg/ml PMA and 1 μg/ml PHA,respectively. After approximately 24 hr, one ml growth medium containing10% FBS, 50 μg/ml PMA and 1 μg/ml PHA was added to each well.Antibiotics were never present during lipid-mediated transfections.Cells were harvested at approximately 48 hr post-transfection bycentrifugation. Cell lysates were prepared by resuspending cell pelletsat 0° C. in 1 M Tris-HCl pH 8.0 containing 0.1% Triton X-100 and 5 μlaliquots were assayed for CAT activity.

Example 9 Transient transfection assays

[0118] Cell lysates were assayed for β-galactosidase activity asdescribed by Sanes et al. (1986) EMBO J. 5:3133. Cells were rinsed withPBS, fixed for 5 minutes in 2% (v/v) formaldehyde, 0.2% glutaraldehydein PBS, rinsed twice with PBS, and stained 2 hr to overnight with 0.1%β-gal, 5 mM potassium ferrocyanide, 5 mM potassium ferrocyanide, 2 mMMgCl₂ in PBS. Rinsed cells were photographed using a 10× or 20×objective on a Nikon inverted microscope with Hoffman optics.Transfection efficiency is evaluated by counting or estimating thenumber of β-gal positive cells (blue-stained cells).

[0119] Cell lysates were assayed for CAT activity as described byNeumann et al. (1987) BioTechniques 5:444. Cells were rinsed with PBSand frozen at −70° C. in 0.5-1.5 ml 0.1% Triton X-100 in 0.1 M Tris, pH8.0. After rapid thawing at 37° C., the lysate was cleared bycentrifugation. When more than 5 μl of a 1 ml extract was to be used inthe assay, the extract was heated to 65° C. for 10 minutes to inactivateany deacetylases present, and centrifuged again. When necessary, theextract was diluted in 0.1 M Tris pH 7.8-8.0. Lysate was incubated with50nCi ¹⁴C Butyryl CoA (New England Nuclear, Boston, Mass.) and 0.25μMoles chloramphenicol in 0.1 M Tris, pH 7.8-8.0, in a total volume of0.25 ml in a 4-ml scintillation vial. Reaction mixtures were incubatedat 37° C. for 2 hr, overlaid with 3 ml Econofluor (Dupont, Boston,Mass.), inverted once and then incubated for an additional 2 hr at roomtemperature before counting. Each assay included a standard curve of0.001 to 0.05 units CAT enzyme (Pharmacia, Uppsala, Sweden), the linearrange for these reaction conditions. Extracts were diluted or volumeswere adjusted in order to have activity within the linear range duringthe assay. To insure accuracy, the activity was normalized to the samevolume per transfection.

Example 10 Results

[0120] Results are shown in Tables 1-4. The “CAT ACTIVITY” column inTable 4 indicates the relative transfection effectiveness of the lipidformulation in Jurkat cells. The control sample was a cell culture grownunder similar conditions as those that were transfected, but with no DNAor cationic lipid added.

[0121] For primary human fibroblast cells (Table 1), NIH-3T3 cells(Table 2), CHO-K1 cells (Table 3), and Jurkat cells (Table 4), compound3 was highly effective for DNA transfection with minimal toxicity. Datain Tables 1-4 show that compound 3 has efficiency comparable to DOSPAfor the transfection of human fibroblasts and Jurkat cells, but a lowerconcentration of lipid is required for optimal activity. When using lowamounts of DNA in NIH-3T3 cells, compound 3 was approximately 10-foldmore effective than DOSPA. TABLE 1 TRANSFECTION RESULTS WITH PRIMARYHUMAN FIBROBLASTS Lipid Optimal Lipid β-gal Positive (Molar ratio) Conc.(μg) Cells (app. %) Compound 3 :DOPE 1 5 (1:1) DOSPA:DOPE (1.5:1) 5 5

[0122] Cells were plated in 24-well plates at a density of 3×10⁴ cellsper well. The following day, cells in each well were transfected with asuboptimal concentration (200 ng) of pCMV β-gal DNA, using the indicatedlipid formulations. TABLE 2 TRANSFECTION RESULTS WITH NIH-3T3 LipidOptimal Lipid β-gal Positive (Molar ratio) Conc. (μg) Cells (app. %)Compound 3:DOPE 1 10 (1:1) DOSPA:DOPE (1.5:1) 4  1

[0123] Cells were plated in 24-well plates at a density of 4×10⁴ cellsper well. The following day, cells in each well were transfected with asuboptimal concentration (200 ng) of pCMV β-gal DNA, using the indicatedlipid formulations. TABLE 3 TRANSFECTION RESULTS WITH CHO-K1 LipidOptimal Lipid β-gal Positive (Molar ratio) Conc. (μg) Cells (app. %)Compound 3:DOPE   1-1.2 20 (1:1) DOTMA:DOPE (1:1) 1.5 10-15 DOSPA:DOPE(1.5:1) 3-5 80-90

[0124] Cells were plated in 24-well plates at a density of 6 ×10⁴ cellsper well. The following day, cells in each well were transfected with200 ng of pCMV β-gal DNA, using the indicated lipid formulations. TABLE4 TRANSFECTION RESULTS WITH JURKAT CELLS Lipid Optimal Lipid CATActivity (Molar ratio) Conc. (μg) (mUnits CAT/5 μl) Compound 3:DOPE 619.8 DOTMA:DOPE (1:1) 5  5.0 DOSPA:DOPE (1.5:1) 25  19.2

[0125] Cells (1×10⁶) were transfected with 2 μg of pCMVCAT DNA asdescribed above, using the indicated lipid formulations. At 48 hrpost-transfection, cell lysates were prepared and 5 μl aliquots wereassayed for CAT enzyme activity.

1. A compound having the formula:

and salts thereof where: X is selected from the group consisting of N,S, P or SO; x is an integer ranging from 1 to about 20; n_(i), where i=1to x, are independently of one another, integers that can have a valueranging from 1 to about 6: r is 0 or 1; such that r is 0 or 1 when X isN with N being positively charged if r is 1, r is 0 when X is S or SOand S and SO are positively charged, and r is 1 when X is P and P ispositively charged; R_(A) and R_(B), independently of one another, areselected from the group consisting of H, or an alkyl, hydroxyalkyl orthiol-substituted alkyl group having from 1 to 6 carbon atoms; R₁ andR₂, independently of other R₁ and R₂, is selected from the groupconsisting of alkyl groups having from 1 to about 6 carbon atoms; A₁ andA₂, independently of other A₁ and A₂ groups, is selected from the groupconsisting of the groups: (a) a straight-chain or branched alkyl,alkenyl, or alkynyl group having from 2 to about 22 carbon atoms whereinone or more non-neighboring —CH₂— groups can be replaced with an O or Satom; (b) straight-chain or branched alkyl group substituted with one ortwo OH, SH, NH₂ or amine groups within about 3 carbon atoms of the bondbetween A₁ or A₂ and X; (c) a substituted straight-chain or branchedalkyl, alkenyl or alkynyl group having from 2 to about 22 carbon atomswherein the substituent is an aromatic, alicyclic heterocyclic orpolycyclic ring and wherein one or more of the non-neighboring —CH₂—groups of said alkyl, alkenyl or alkynyl group can be substituted withan O or S atom; (d) a —B—L group where B is selected from the groupconsisting of —CO—, —CO₂—, —OCO—, —CO—NH—. —O—CO—NH—, —O—CH₂—,—CH₂—O—,—S—CH₂—, —CH₂—S—, and —CH₂— and L is selected from the group consistingof those groups defined in (a) or (c), above, an aromatic, alicyclic,heterocyclic, and a polycyclic ring moiety; (e) a —CH(D—L)₂ or a—C(D—L)₃ group where D is selected from the group consisting of —CO—,—CO₂—, —OCO—, —CO—NH—, —O—CO—NH—, —O—, and —S— and L is selected fromthe group consisting of those groups defined in (a) or (c), above, anaromatic, alicyclic, heterocyclic, and polycyclic ring moiety.
 2. Thecompound according to claim 1 wherein X is N, r is 1 and A₁ and A₂,independently of one another, are selected from the group consisting ofa straight-chain or branched alkyl, alkenyl, or alkynyl group havingfrom 2 to about 22 carbon atoms wherein one or more non-neighboring—CH₂— groups is replaced with an O or S atom.
 3. The compound accordingto claim 1 wherein X is N, r is 1 and A₁ and A₂, independently of oneanother, are selected from the group consisting of a straight-chain orbranched alkyl group substituted with one or two SH, NH₂ or amine groupswithin about 3 carbon atoms of the bond between A₁ or A₂ and X.
 4. Thecompound according to claim 1 wherein X is N and A₁ and A₂,independently of one another, are selected from the group consisting ofa substituted straight-chain or branched alkyl, alkenyl or alkynyl grouphaving from 2 to about 22 carbon atoms wherein the substituent is anaromatic, alicyclic heterocyclic or polycyclic ring and wherein one ormore of the non-neighboring —CH₂— groups of said alkyl, alkenyl oralkynyl group can be substituted with an O or S atom.
 5. The compound ofclaim 1 wherein X is N and A₁ and A₂, independently of one another, areselected from the group consisting of a —B—L group where B is selectedfrom the group consisting of —CO—, —CO₂—, —OCO—, —CO—NH—. —O—CO—NH—,—O—CH₂—, —CH₂—O—, —S—CH₂—, —CH₂—S—, and —CH₂— and L is selected from thegroup consisting of those groups defined in (a) or (c), above, anaromatic, alicyclic, heterocyclic, and a polycyclic ring moiety.
 6. Thecompound of claim 5 wherein B is —O—CH₂—.
 7. The compound of claim 5wherein B is —CO—N— or —O—CO—N—.
 8. The compound of claim 5 wherein L isa straight-chain or branched alkyl, alkenyl, or alkynyl group havingfrom 2 to about 22 carbon atoms wherein one or more non-neighboring—CH₂— groups can be replaced with an O or S atom.
 9. The compound ofclaim 1 wherein X is N and wherein A₁ and A₂ are selected from the groupconsisting of an —CH(D-L)₂ or a —C(D-L)₃ group where D is selected fromthe group consisting of —CO—, —CO₂—, —OCO—, —CO—NH—. —O—CO—NH—, —O—, and—S— and L is selected from the group consisting of those groups definedin (a) or (c), above, an aromatic, alicyclic, heterocyclic, andpolycyclic ring moiety.
 10. The compound of claim 9 wherein D isselected from the group consisting of —O—, —CO—, —OCO—, and —CO₂—. 11.The compound of claim 9 wherein L is a straight-chain or branched alkyl,alkenyl, or alkynyl group having from 2 to about 22 carbon atoms whereinone or more non-neighboring —CH₂— groups can be replaced with an O or Satom.
 12. A lipid aggregate which comprises one or more compounds ofclaim
 1. 13. A lipid aggregate of claim 12 further comprising one ormore phospholipids.
 14. A composition for transfecting a cell whichcomprises a compound of claim 1 and a nucleic acid.
 15. A compositionfor transfecting a cell of claim 14 further comprising one or moreamphipathic lipids.
 16. A composition for transfecting a cell of claim14 further comprising a phospholipid.
 17. A composition for transfectinga cell which comprises a lipid aggregate of claim 12 and a nucleic acid.18. A kit for preparing a lipid aggregate comprising one or morecompounds of claim
 1. 19. The kit of claim 18 further comprising one ormore phospholipids.
 20. A method for transfecting a cell comprising thestep of contacting said cell with a lipid aggregate comprising a nucleicacid and a compound of claim
 1. 21. A method for transfecting a cellcomprising the step of contacting said cell with the composition ofclaim
 14. 22. A method for delivery of a macromolecule to a cellcomprising the step of contacting said cell with a composition whichcomprises a compound of claim 1 and said macromolecule.
 23. The methodof claim 22 wherein the macromolecule is an anionic molecule.
 24. Acompound having the formula

and salts thereof where: X is selected from the group consisting of N,S, P or SO; m and n, independently of one another, are integers rangingfrom 1 to about 6; x is an integer ranging from 1 to about 10; r is 0 or1; such that r is 0 or 1 when X is N with N being positively charged ifr is 1, r is 0 when X is S or SO and S and SO are positively charged,and r is 1 when X is P and P is positively charged; R_(A) and R_(B),independently of one another, are selected from the group consisting ofH, or an alkyl hydroxyalkyl or thiol substituted alkyl group having from1 to about 6 carbon atoms; R₁-R₃, independently of one another, areselected from the group consisting of alkyl groups having from 1 toabout 6 carbon atoms, where r is either 1 or 0, such that r is 0 or 1when X is N, r is 0 when X is S or SO, and r is 1 when X is P; and A₁,A₂, and A₃, independently of other A₁, A₂ and A₃ groups, is selectedfrom the group consisting of the groups: (a) a straight-chain orbranched alkyl, alkenyl, or alkynyl group having from 2 to about 22carbon atoms wherein one or more non-neighboring —CH₂— groups can bereplaced with an O or S atom; (b) a straight-chain or branched alkylgroup substituted with one or two OH, SH, NH₂ or amine groups withinabout 3 carbon atoms of the bond between A₁ or A₂ and X; (c) asubstituted straight-chain or branched alkyl, alkenyl or alkynyl grouphaving from 2 to about 22 carbon atoms wherein the substituent is anaromatic, alicyclic heterocyclic or polycyclic ring and wherein one ormore of the non-neighboring —CH₂— groups of said alkyl, alkenyl oralkynyl group can be substituted with an O or S atom; (d) a —B—L groupwhere B is selected from the group consisting of —CO—, —CO₂—, —OCO—,—CO—NH—, —O—CO—NH—, —O—CH₂—, —CH₂—O—, —S—CH₂—, —CH₂—S—, and —CH₂— and Lis selected from the group consisting of those groups defined in (a) or(c), above, an aromatic, alicyclic, heterocyclic, and a polycyclic ringmoiety; (e) a —CH(D—L)₂ or a —C(D—L)₃ group where D is selected from thegroup consisting of —CO—, —CO₂—, —OCO—, —CO—NH—, —O—CO—NH—, —O—, and —S—and L is selected from the group consisting of those groups defined in(a) or (c), above, an aromatic, alicyclic, heterocyclic, and polycyclicring moiety.
 25. The compound of claim 24 wherein A₁ and A₂,independently of one another, are selected from the group consisting ofstraight chain alkyl, alkenyl, or alkynyl groups.
 26. A compound havingthe formula

and salts thereof where: X is selected from the group consisting of N,S, P or SO; l, m and n, independently of one another, are integersranging from 1 to about 6; x is an integer ranging from 1 to about 10; ris 0 or 1; such that r is 0 or 1 when X is N with N being positivelycharged if r is 1, r is 0 when X is S or SO and S and SO are positivelycharged, and r is 1 when X is P and P is positively charged; R_(A) andR_(B), independently of one another, are selected from the groupconsisting of H, or an alkyl, hydroxyalkyl or thiol substituted alkylgroup having from 1 to about 6 carbon atoms; R₁-R₄, independently of oneanother, are selected from the group consisting of alkyl groups havingfrom 1 to about 6 carbon atoms, where r is either 1 or 0, such that r is0 or 1 when X is N, r is O when X is S or SO, and r is 1 when X is P;and A₁, A₂, A₃ and A4, independently of other A₁, A₂, A₃ and A4 groups,is selected from the group consisting of the groups: (a) astraight-chain or branched alkyl, alkenyl, or alkynyl group having from2 to about 22 carbon atoms wherein one or more non-neighboring —CH₂—groups can be replaced with an O or S atom; (b) a straight-chain orbranched alkyl group substituted with one or two OH, SH, NH2 or aminegroups within about 3 carbon atoms of the bond between A₁or A₂ and X;(c) a substituted straight-chain or branched alkyl, alkenyl or alkynylgroup having from 2 to about 22 carbon atoms wherein the substituent isan aromatic, alicyclic heterocyclic or polycyclic ring and wherein oneor more of the non-neighboring —CH₂— groups of said alkyl, alkenyl oralkynyl group can be substituted with an O or S atom; (d) a —B—L groupwhere B is selected from the group consisting of —CO—, —CO₂—, —OCO—,—CO—NH—, —O—CO—NH—, —O—CH₂—, —CH₂—O—, —S—CH₂—, —CH₂—S—, and —CH₂— and Lis selected from the group consisting of those groups defined in (a) or(c), above, an aromatic, alicyclic, heterocyclic, and a polycyclic ringmoiety; (e) a —CH(D—L)₂ or a —C(D—L)₃ group where D is selected from thegroup consisting of —CO—, —CO₂—, —OCO—, —CO—NH—, —O—CO—NH—, —O—, and —S—and L is selected from the group consisting of those groups defined in(a) or (c), above, an aromatic, alicyclic, heterocyclic, and polycyclicring moiety.
 27. The compound of claim 1 having the structure wherein nis an integer between 2 and 6 inclusive, and R is an unbranched alkyl,alkenyl, alkynyl or alkoxy having 2 to 22 carbon atoms.


28. The compound

wherein Ar₁ and Ar₂ are aryl rings, and R is selected from any of R₅-R₈where R₅ is an unbranched alkyl, alkenyl, alkynyl or alkoxy having 2 to22 carbon atoms; or R₆ is a branched alkyl, alkenyl, alkynyl or alkoxy,wherein each branch has 2 to 22 carbon atoms; or R₇ is substituted orunsubstituted aromatic, alicyclic, heterocyclic or polycyclic; or R₈ isbranched or unbranched substituted alkyl, alkenyl, alkynyl or alkoxyhaving 2 to 22 carbon atoms, wherein the substituent is a substituted orunsubstituted aromatic, alicyclic, heterocyclic or polycyclic ring. 29.A lipid aggregate which comprises one or more compounds of claim
 28. 30.A method for transfecting a cell comprising the step of contacting saidcell with a lipid aggregate comprising a nucleic acid and a compound ofclaim 28.